I am getting a very low recovery by Native ChIP for H3 using our lab's antibody that work great in X-ChIP (I measure it with qPCR and get values around 0.1%, although in X-ChIP the same primers give 10-30% recovery). I am using chromatin from 5*10^6 cells which I also tried to dilute 1:50 as I read that it can increase ChIP specificity. The same protocol for native ChIP works absolutely fine for various histone modifications. Any suggestions why it is not working and what I can try to improve?
Some possibilities:
1. Some cells have inherently lower efficiency for ChIP-Seq eg muscle and adipose
2. Ab is not working efficiently. Too many freeze thaws can do this.
3. Presumably the fragmentation of chromatin is different> sonication vs MNase digestion. This could be an issue. Are you starting with the same amount of soluble chromatin? Perhaps run the purified DNA from the soluble chromatin starting material on a gel to ensure that you have the correct size range.
Thank you for your suggestions! I tried different antibodies and they worked. As I see it - some antibodies just simply don't work in Native ChIP although they work in X-ChIP. It might be something to do with their antigen accessibility in different conditions of ChIP
If the antibody works for X-ChIP, and in western blots, etc..the problem should be theMNsase treatment; try to reduce it ( play with different times/enzyme concetrations...)
On the other hand, maybe it is easy to try another H3 antibody from another supplier.
It is all about epitope exposure-- most general H3 Abs are raised against peptide epitopes on the C-terminus, most of which require the nucleosome to unfold (sonication does this to some extent) in order for them to be able to bind efficiently.
Out of curiosity, which Antibody ultimately worked?
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